The invention relates to expression systems for producing collagen; more particularly, the invention relates to co-expression of collagen and prolyl 4-hydroxylase in stably transfected insect cells.
Collagens are extracellular matrix proteins that contain the repeating triplet sequence Gly-X-Y and the presence of such triplets allows three collagen polypeptide chains (α-chains) to fold into a triple-helical conformation. The Y position amino acid in the triplet sequence Gly-X-Y is frequently proline, which is often 4-hydroxylated by post-translational modification of the collagen polypeptide chain in order to stabilize the triple-helical structure of collagen. In the absence of proline hydroxylation, the essential triple helical conformation of collagen is thermally unstable at below physiological temperatures (Berg, R. A., and Prockop, D. J. (1973) Biochem Biophys Res Commun 52, 115-120; Rosenbloom, J., et al. (1973) Arch Biochem Biophys 158, 478-484). Prolyl 4-hydroxylase (EC 1.14.11.2), the key enzyme catalyzing the 4-hydroxylation of proline residues in all collagens, from vertebrates is an α2β2 tetramer consisting of two different types of subunits (Kivirikko, K. I., et al., (1989) Faseb J3, 1609-1617). The α subunit contains the catalytic and peptide-substrate binding domains but is inactive in the absence of the β subunit. The β subunit was found to be identical to the enzyme protein-disulfide isomerase (Koivu, J., et al. (1987) J Biol Chem 262, 6447-6449; and Pihlajaniemi, T., et al. (1987) Embo J6, 643-649). During collagen biosynthesis, the procollagen α chains are co-translationally transported into the lumen of the endoplasmic reticulum where they are hydroxylated by prolyl 4-hydroxylase. Prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate, and an active system appears to exist in vertebrate cells for the transport of 2-oxoglutarate and ascorbate into the lumen of the endoplasmic reticulum (Kivirikko, K. I., et al. (1989) Faseb J3, 1609-1617). In vitro expression of an active recombinant prolyl 4-hydroxylase from its subunits has been successfully obtained by co-infection of insect cells Spodoptera frugiperda and Trichoplusia ni (Vuori, K., et al. (1992) Proc Natl Acad Sci USA 89, 7467-7470) with recombinant baculoviruses, or cotransfection with expression vectors in mammalian cell lines COS-1 (John, D. C., and Bulleid, N. J. (1996) Biochem J 317 (Pt 3), 659-665) and HEK293 (Wagner, K., et al. (2000) Biochem J352 Pt 3, 907-911), in yeasts Pichia pastoris (Vuorela, A., et al. (1997) Embo J16, 6702-6712) and Saccharomyces cerevisiae (Toman, P. D., et al. (2000) J Biol Chem 275, 23303-23309). The prolyl 4-hydroxylase tetramer assembly probably requires molecular chaperones, such as immunoglobulin heavy chain binding protein, BiP (John, D. C., and Bulleid, N. J. (1996) Biochem J 317 (Pt 3), 659-665; Veijola, J., et al. (1996) Biochem J315 (Pt 2), 613-618).
Expression systems for producing recombinant collagens in the present includes E. coli, yeast, mammalian cell lines, insect cells, as well as transgenic animals and plants, however, these expression systems have their own disadvantages. For example, E. coli expression system has no post-translational modification, and yeast expression system lacks prolyl 4-hydroxylase activity, through the yield of collagens in Pichia pastoris was the highest among these expression systems. Mammalian cell line expression system has low yield and limits to specific tissue types, and insect cell expression system has low prolyl 4-hydroxylase activity. As for transgenic animals, such as silk worm or mice, or plant, such as tobacco, the collagen products were overly cross-linked. A baculovirus expression system containing genes encoding human prolyl 4-hydroxylase and collagen has been established (U.S. Pat. Nos. 5,077,214 and 5,593,859), however, the transfected insect cells will be destroyed in 72 hours, resulting in only transient recombinant protein production. In addition, it is difficult to recover or purify the recombinant collagens because of cell lysis. Moreover, collagens are secreted or membrane proteins, and the destruction of endoplasmic reticulum or Golgi body by the baculovirus limits the production of collagens. An expression system for producing biologically active collagens in high yield is, therefore, still required.